Around 46% of the World's population lives in areas where mosquito-borne diseases including malaria, filariasis, viral encephalitides, dengue and yellow fever are endemic. It was recently established that the insect pathogenic fungus Metarhizium anisopliae has the potential to control adult mosquitoes in an urban setting, but only if its potency is increased. Our goal is to produce one or more fungal products which can deplete anopheline and Plasmodium populations to the extent that marked reductions in malaria prevalence are achieved. We have already shown that M. anisopliae is a very effective delivery system for the insect-selective scorpion toxin AaIT, and that expressing AaIT produced a 9-fold reduction in effective spore doses against mosquitoes. This was very significant but kill times remain too slow for adequate protection. In this application, we propose experiments to compare several strategies for optimizing M. anisopliae's ability to curtail disease transmission. Mosquitoes infected with fungi showed a significant reduction in the number of sporozoites on salivary glands, but the mechanism responsible is unknown. We will carry out a detailed analysis of the interactions between Plasmodium, mosquitoes and M. anisopliae. This will include testing an attenuated strain of M. anisopliae that elicits a hyperimmune response to determine whether M. anisopliae can be used to immunize mosquitoes from Plasmodium. Further, we will compare mortality and sporozoite prevalence in mosquitoes infected with M. anisopliae strains expressing different combinations of insecticidal and anti-plasmodial proteins. It will be determined if these can be used synergistically to achieve effective reductions in transmission potential. Based on these results, we will also test the efficacy of using M. anisopliae to express synthetic multifunctional genes that are hybrids of different activities and that could, for example, target both the insect and the Plasmodium. The current proposal will: 1) explore the mosquito immune system;2) develop tools and genetically engineered fungi that have the potential to greatly reduce malaria prevalence, and 3) develop M. anisopliae as a tractable model system that can be used to screen novel effectors. We envisage that after screening, the most potent effectors could be delivered against mosquitoes or Plasmodium by expression in M. anisopliae and/or in alternative pathogens, commensals or via transgenic mosquitoes. PUBLIC HEALTH RELEVANCE This project aims to design, construct and evaluate recombinant fungal pathogens that target adult mosquitoes and the malaria parasite. The most significant possible outcome of producing an optimized fungal pathogen will be a reduction of human disease as a result of interrupting transmission of the target parasite.